Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• This invention relates to novel humanized monoclonal antibodies (mAbs) and to the genes encoding same. More specifically, this invention relates to human monoclonal antibodies specifically reactive with several heterodimeric integrins. Such antibodies are useful for the therapeutic and/or prophylactic treatment of a variety of angiogenic associated diseases (e.g., cancer metastasis, rheumatoid arthritis, atherosclerosis) among other disorders.
• angiogenic associated diseases e.g., cancer metastasis, rheumatoid arthritis, atherosclerosis
• Integrins are a superfamily of cell adhesion receptors, which are heterodimeric transmembrane glycoproteins expressed on a variety of cells, and which are made up of a variety of alpha and beta chains. Among those integrins are those which carry the RGD ligand, wherein one protein chain is referred to as ⁇ .
• the integrins or cell surface adhesion receptors which share the ⁇ v chain include the vitronectin receptor a v ⁇ ,. expressed on a number of cells, including endothelial, smooth muscle, osteoclast, and tumor cells; and also the ⁇ v ⁇ 5 . c. v ⁇ 6, -v ⁇ anc ⁇ a ⁇ receptors. These receptors are expressed primarily on cancer cells, such as tumor cells and in metastatic lesions. They are present on some immunohistologic lesions, in the placenta and are also involved angiogenic disorders involving the vasculature.
• the ⁇ v ⁇ 3 receptor expressed on the membrane of osteoclast cells has been postulated to mediate the bone resorption process and contribute to the development of osteoporosis [Ross, et ai, J. Biol. Chem., 1993, 268 (13): 9901 -7].
• the v ⁇ 3 receptor expressed on human aortic smooth muscle cells has been postulated to stimulate their migration into neointima, which leads to the formation of atherosclerosis and restenosis after angioplasty [Brown, et al., Cardiovascular Res., 1994, 28: 1815].
• a recent study referred to an 0 ⁇ . 3 antagonist as promoting tumor regression by inducing apoptosis of angiogenic blood vessels [P. C. Brooks, et al., Cell, 1994, 79: 1157-1164].
• a murine monoclonal antibody LM609 developed to the vitronectin receptor reported in International Patent Publication No. WO89/05155, published June 15, 1995, was referred to as useful in the inhibition of tumor growth. See, also, D. A. Cheresh et al, Cell, 1989, 57:59-69.
• CDRs Complementarity Determining Regions
• Novel human mABs or humanized antibodies are particularly useful alone or in combination with existing molecules to form immunotherapeutic compositions. There remains a need in the art for additional mAbs to cell surface receptors or humanized antibodies thereto which can selectively block the integrin and display a long serum half-life.
• this invention relates to a novel humanized monoclonal antibody which neutralizes the biological function of receptors which carry the v chain.
• this antibody is reactive with a variety of such heterodimeric integrins and functional fragments thereof which carry the v subunit chain.
• This humanized antibody specifically neutralizes the human v ⁇ (vitronectin receptor) and the human v ⁇ 5 receptor.
• the present invention provides modifications to neutralizing Fab fragments or F(ab') ⁇ fragments specific for the human ⁇ v B 3 and v ⁇ 5 receptors produced by random combinatorial cloning of human antibody sequences and isolated from a filamentous phage Fab display library.
• a reshaped human antibody containing human heavy and light chain constant regions from a first human donor and heavy and light chain variable regions or the CDRs thereof derived from human neutralizing monoclonal antibodies for the human v ⁇ 3 / v ⁇ 5 receptors derived from a second human donor.
• the present invention provides a pharmaceutical composition which contains one (or more) altered antibodies and a pharmaceutically acceptable carrier.
• the present invention provides a method for passive immunotherapy of a disorder mediated by a receptor carrying the ⁇ v subunit, such as all cancers characterized by solid tumors that metastasize, lymphomas that express these receptors; angiogenesis of the target of the tumors, restenosis, rheumatoid arthritis or atherosclerosis, among others, in a human by administering to said human an effective amount of the pharmaceutical composition of the invention for the prophylactic or therapeutic treatment of the disorder.
• the invention provides a method for treating a disease which is mediated by an v -containing heterodimeric integrin in a human, by administering to the human an immunotherapeutically effective amount of the antibody of the invention, followed by administering to said human a therapeutically effective amount of a small chemical molecule which is an antagonist of the integrin receptor.
• These antibodies may also be co- administered with other drugs which are known for the treatment of the particular cancer or angiogenic disorder.
• the present invention provides methods for, and components useful in, the recombinant production of humanized and altered antibodies (e.g., engineered antibodies, CDRs, Fab or F(ab) 2 fragments, or analogs thereof) which are derived from neutralizing monoclonal antibodies (mAbs) for the human v -containing receptors.
• These components include isolated nucleic acid sequences encoding same, as well as recombinant plasmids containing the nucleic acid sequences under the control of selected regulatory sequences which are capable of directing the expression thereof in host cells (preferably mammalian) transfected with the recombinant plasmids.
• the production method involves culturing a transfected host cell of the present invention under conditions such that the human or altered antibody is expressed in said cell and isolating the expressed product therefrom.
• Yet another aspect of the invention is a method to diagnose the overexpression of the human ct v -containing receptor in a human.
• the method includes contacting a biopsy sample with the antibodies and altered antibodies of the instant invention and assaying for the occurrence of binding between said antibody (or altered antibody) and the human c v - containing heterodimeric receptor using a conventional imaging system.
• Yet another embodiment of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising at least one dose of an immunotherapeutically effective amount of the antibodies of this invention in combination with at least one additional monoclonal or altered antibody.
• a particularly desirable composition comprises as the additional antibody, an anti-human ⁇ receptor antibody, which is distinguished from the subject antibody by virtue of being reactive with a different epitope of the heterodimeric human a v B s receptor.
• an anti-human ⁇ receptor antibody which is distinguished from the subject antibody by virtue of being reactive with a different epitope of the heterodimeric human a v B s receptor.
• One such example is the D12 antibody described in International Patent Application No. WO98/40488, published September 17, 1998.
• Fig. 1 A illustrates the heavy chain variable region DNA and amino acid sequences of the murine mAb B9 [SEQ ID NOS: 1 and 2], in which the CDRs are represented in bold and underlined.
• Fig. IB is the heavy chain variable region amino acid sequence of the human VH subgroup I consensus amino acid sequence [SEQ ID NO: 3], in which the CDRs are represented in bold and underlined.
• Fig. 1 C illustrates the synthetic heavy chain variable region DNA and amino acid sequences of the consensus, humanized heavy chain B9HZHC 1 -0 [SEQ ID NOS: 4 and 5], in which the CDRs are represented in bold and underlined. Asterisks above the amino acids indicate preferred murine framework residues retained in the synthetic heavy chain.
• Fig. 2A illustrates the light chain DNA and amino acid sequences of the murine mAb B9 [SEQ ID NOS: 6 and 7], in which the CDRs are represented in bold and underlined.
• Fig. 2B is the light chain amino acid sequence of the human V kappa subgroup I consensus amino acid sequence of [SEQ ID NO: 8], in which the CDRs are represented in bold and underlined.
• Fig. 2C is the synthetic light chain amino acid DNA and amino acid sequence of the consensus, synthetic, humanized light chain B9HZLC1 -0 [SEQ ID NOS: 9 and 10], in which the CDRs are represented in bold and underlined. Asterisks indicate preferred murine framework residues and are illustrated above the amino acid residue.
• Fig. 3 illustrates the DNA and amino acid sequences representing the intermediate of the synthetic B9 heavy chain variable region [SEQ ID NOS: 1 1 and 12].
• the endonuclease enzyme restriction sites are underlined.
• Fig. 4 illustrates the DNA and amino acid sequences representing the intermediate of the synthetic B9 light chain variable region [SEQ ID NOS: 13 and 14].
• the endonuclease enzyme restriction sites are underlined.
• Fig. 5 is the amino acid sequence of the modified human REI kappa V region framework with the CDRs underlined and in bold type.
• the last QQ of the sequence represents the first two amino acids of the third CDR [SEQ ID NO: 15].
• Fig. 6 illustrates the DNA and amino acid sequences [SEQ ID NOS: 28 and 29] representing a light chain encoding sequence of humanized B9 AB9HZLCREI__. The CDRs are shown in bold print.
• Fig. 7 illustrates the DNA and amino acid sequences [SEQ ID NOS: 30 and 31] of the light chain encoding sequence of the humanized B9, AB9HZLC1-1__.
• Fig. 8 is a graph showing the results of the mouse tumor xenograft model with HT29 assay of Example 14, with the cells injected i.p.
• Fig. 9 is a graph showing the results of the mouse tumor xenograft model with HT29 assay of Example 14, with the cells injected s.c.
• the present invention provides useful antibodies, including monoclonal, recombinant and synthetic antibodies (and fragments thereof) which neutralize the human vitronectin ⁇ v B receptor and the human ⁇ v ⁇ ; receptors, as well as bind to other integnns bearing the oc v subunit, isolated nucleic acids encoding same and vanous means for their recombinant production as well as therapeutic, prophylactic and diagnostic uses of such antibodies and fragments thereof
• the antibodies of this invention inhibit the binding of vitronectin and other ligands to the vitronectin ( ⁇ v B ) receptor
• the antibodies of this invention inhibit the binding of ligands to the cc v ⁇ i receptor
• These antibodies may also inhibit the binding of ligands to the ⁇ v ⁇ and ⁇ v ⁇ 6 and v ⁇ 8 receptors
• These antibodies can selectively block the integrins ⁇ v B and ⁇ v ⁇ and display a serum half-life of approximately 60 hours in vivo in animal models
• the antibodies including the munne monoclonal B9 and the humanized antibody HuB9, which specifically neutralize ⁇ v B3 and oc v ⁇ ; are desirable for use as acute and subacute therapeutic reagents for the treatment of the disorders mediated by these receptors Inhibition of these receptors by the antibodies of this invention permits therapeutic treatment or prophylaxis of such diseases
• the phrase "disorders mediated by v -conta ⁇ n ⁇ ng heterodimenc receptors" includes, but is not limited to, cardiovascular disorders or angiogenic-related disorders, such as cancers, e g , solid tumor and metastases, angiogenesis associated with diabetic retinopathy, atherosclerosis and restenosis, chronic inflammatory disorders, macular degeneration, and diseases wherein bone resorption is associated with pathology such as osteoporosis
• the antibodies of this invention are p ⁇ manly useful also as anti-metastatic and antitumor agents
• Altered antibody refers to a protein encoded by an immunoglobulin coding region altered from its natural form, which may be obtained by expression in a selected host cell Such altered antibodies are engineered antibodies (e g , chime ⁇ c, humanized, or reshaped or immunologically edited human antibodies) or fragments thereof lacking all or part of an immunoglobulin constant region, e g , Fv, Fab, or F(ab')
• altered immunoglobulin coding region refers to a nucleic acid sequence encoding an altered antibody of the invention or a fragment thereof
• Reshaped human antibody refers to an altered antibody in which minimally at least one CDR from a first human monoclonal donor antibody is substituted for a CDR in a second human acceptor antibody
• all six CDRs are replaced More preferably an entire antigen combining region, for example, an Fv, Fab or F(ab') 2 , from a first human donor monoclonal antibody is substituted for the corresponding region in a second human acceptor monoclonal antibody.
• the Fab region from a first human donor is operatively linked to the appropriate constant regions of a second human acceptor antibody to form a full length monoclonal antibody.
• First immunoglobulin partner refers to a nucleic acid sequence encoding a human framework or human immunoglobulin variable region in which the native (or naturally- occurring) CDR-encoding regions are replaced by the CDR-encoding regions of a donor human antibody.
• the human variable region can be an immunoglobulin heavy chain, a light chain (or both chains), an analog or functional fragment thereof.
• Such CDR-encoding regions, located within the variable region of antibodies (immunoglobulins) can be determined by known methods in the art. For example Kabat et al, Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987) disclose rules for locating CDRs.
• “Second fusion partner” refers to another nucleotide sequence encoding a protein or peptide to which the first immunoglobulin partner is fused in frame or by means of an optional conventional linker sequence (i.e., operatively linked).
• the fusion partner is an immunoglobulin gene and when so, it is referred to as a "second immunoglobulin partner".
• the second immunoglobulin partner may include a nucleic acid sequence encoding the entire constant region for the same (i.e., homologous - the first and second altered antibodies are derived from the same source) or an additional (i.e., heterologous) antibody of interest.
• the second immunoglobulin partner may be an immunoglobulin heavy chain or light chain, or both chains as part of a single polypeptide.
• the second immunoglobulin partner is not limited to a particular immunoglobulin class or isotype.
• the second immunoglobulin partner may comprise part of an immunoglobulin constant region, such as found in a Fab, or F(ab) 2 (i.e., a discrete part of an appropriate human constant region or framework region).
• a second fusion partner may also comprise a sequence encoding an integral membrane protein exposed on the outer surface of a host cell, e.g., as part of a phage display library, or a sequence encoding a protein for analytical or diagnostic detection, e.g., horseradish peroxidase, ⁇ -galactosidase, etc.
• Fv, Fc, Fd, Fab, or F(ab') 2 are used with their standard meanings [see, e.g., Harlow et al, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)].
• an “engineered antibody” describes a type of altered antibody, i.e., a full-length synthetic antibody (e.g., a chimeric, humanized, reshaped or immunologically edited human antibody as opposed to an antibody fragment) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope.
• a full-length synthetic antibody e.g., a chimeric, humanized, reshaped or immunologically edited human antibody as opposed to an antibody fragment
• a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies which have specificity for the selected epitope.
• such molecules may include antibodies characterized by a humanized heavy chain associated with an unmodified light chain (or chimeric light chain), or vice versa.
• Engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain framework regions in order to retain donor antibody binding specificity. These antibodies can comprise replacement of one or more CDRs (preferably all) from the acceptor antibody with CDRs from a donor antibody described herein.
• a “chimeric antibody” refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy chain) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody from a heterologous species.
• a “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
• framework support residues may be altered to preserve binding affinity [see, e.g., Queen et al., 1991, Proc. Natl Acad. Sci. USA, 86: 10029-10032 and Hodgson et ai, 1991, Bio/Technology, 9:421].
• an "immunologically edited antibody” refers to a type of engineered antibody in which changes are made in donor and/or acceptor sequences to edit regions involving cloning artifacts, germ line enhancements, etc. aimed at reducing the likelihood of an immunological response to the antibody on the part of a patient being treated with the edited antibody.
• donor antibody refers to an antibody (monoclonal, or recombinant) which contributes the nucleic acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
• One donor antibody suitable for use in this invention is a neutralizing murine monoclonal anti- ( v B_ and anti-ci v ⁇ s antibody, designated as B9.
• B9 is defined as a having the variable heavy chain and variable light chain amino acid sequences shown in Figs. 1 A and 2A, respectively [SEQ ID NOS: 2 and 7].
• acceptor antibody refers to an antibody (monoclonal, or recombinant) from a source genetically unrelated to the donor antibody, which contributes all (or any portion, but preferably all) of the nucleic acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
• a human antibody is the acceptor antibody.
• Consensus VH or “Consensus VL” regions refers to amino acid sequences which can function in a manner similar to the framework regions of the acceptor antibody, but are selected by conventional computer techniques. Briefly, provided with a given VH or VL amino acid sequence, the human VH and VL sequences closest to the given sequence are assembled to identify the closest antibody subgroup. Once the subgroup is selected, all human antibodies from that subgroup are compared and a consensus sequence of the VH and VL chains are prepared. The consensus sequences are used to generate a desirable synthetic framework region for the humanized antibody.
• CDRs are the complementarity determining region amino acid sequences of an antibody. CDRs are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al, Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portions of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate).
• CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
• CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
• mAb B9 may be characterized by a certain level of antigen affinity, a CDR encoded by a nucleic acid sequence of mAb B9 in an appropriate structural environment may have a lower, or higher affinity. It is expected that CDRs of mAb B9 in such environments will nevertheless recognize the same epitope(s) as does the intact mAb B9.
• a "functional fragment” is a partial heavy or light chain variable sequence (e.g., minor deletions at the amino or carboxy terminus of the immunoglobulin variable region) which retains the same antigen binding specificity and/or neutralizing ability as the antibody from which the fragment was derived.
• an “analog” is an amino acid sequence modified by at least one amino acid, wherein said modification can be a chemical modification or substitution onto an amino acid or a substitution or a rearrangement of a few amino acids (i.e., no more than 10), which modification permits the amino acid sequence to retain the biological characteristics, e.g., antigen specificity and high affinity, of the unmodified sequence.
• different humanized variants can be constructed using the sequences disclosed herein.
• Analogs may also arise as allelic variations.
• An "allelic variation or modification” is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention. Such variations or modifications may be due to degeneracy in the genetic code or may be deliberately engineered to provide desired characteristics.
• effector agents refers to non-protein carrier molecules to which the altered antibodies, and/or natural or synthetic light or heavy chains of the donor antibody or other fragments of the donor antibody may be associated by conventional means.
• non-protein carriers can include conventional carriers used in the diagnostic field, e.g., polystyrene or other plastic beads, polysaccharides, e.g., as used in the BIAcore (Pharmacia) system, or other non-protein substances useful in the medical field and safe for administration to humans and animals.
• Other effector agents may include a macrocycle, for chelating a heavy metal atom, or radioisotopes. Such effector agents may also be useful to increase the half-life of the altered antibodies, e.g., polyethylene glycol.
• a non-human species may be employed to generate a desirable immunoglobulin upon presentment with a human ⁇ v - containing heterodimeric receptor as antigen.
• Conventional hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human monoclonal antibody (mAb) to the ⁇ v -containing receptors.
• mAb monoclonal antibody
• the production of murine mAb B9 is described in detail in Example 2 below.
• the term B9 may refer to the B9 mAb or any of the other mAbs of Example 2.
• the B9 is a desirable donor antibody for use in developing a chimeric or humanized antibody of this invention.
• the characteristics of the neutralizing murine mAb B9 obtained as described in Example 2 include an antigen binding specificity for human v B 3 and ⁇ v ⁇ 5 , and possibly other v -containing heterodimeric receptors, and characteristics listed in Table I below.
• the isotype of the mAb B9 of Example 2 is IgG
• the antibody recognizes the ⁇ v subunit of the heterodimeric v ⁇ 3 and v ⁇ 5 receptors and does not recognize either ⁇ subunit individually.
• the binding is illustrated by binding and functional activity (neutralization) in the in vitro assays of Examples 3-10 below.
• B9 the light chain variable region of B9 [SEQ ID NO: 6] and the heavy chain variable region of B9 [SEQ ID NO: 1]
• one of skill in the art could obtain the remaining portions of the heavy chain using, for example, polymerase chain reaction, and thus obtain a complete mAb molecule.
• a B9 molecule could be constructed using techniques analogous to those described below for the synthetic and recombinant mAbs of the invention and employing other murine IgG subtype heavy chains.
• anti-0Cv subunit antibodies may be developed by screening hybridomas or combinatorial libraries, or antibody phage displays [W. D. Huse et al, 1988, Science, 246:1275-1281] using the murine mAb described herein and its ⁇ v epitope.
• a collection of antibodies, including hybridoma products or antibodies derived from any species immunoglobulin repertoire may be screened in a conventional competition assay, such as described in Examples 5, 8 and 9 below, with one or more epitopes described herein.
• the invention may provide an antibody, other than B9, which is capable of binding to and neutralizing the v -containing receptors.
• Other Abs generated against a desired v or suitable ⁇ chain epitope and produced by conventional techniques include without limitation, genes encoding murine mAbs, human mAbs, and combinatorial antibodies.
• This invention is not limited to the use of the B9 mAb or its hypervariable sequences. It is anticipated that any appropriate v ⁇ 3 or v ⁇ 5 neutralizing antibodies and corresponding anti- ⁇ v CDRs described in the art may be substituted therefor. Wherever in the following description the donor antibody is identified as B9, this designation is made for illustration and simplicity of description only.
• the goal of combinatorial cloning is to transfer to a population of bacterial cells the immunological genetic capacity of a human cell, tissue or organ. It is preferred to employ cells, tissues or organs which are immunocompetent. Particularly useful sources include, without limitation, spleen, thymus, lymph nodes, bone marrow, tonsil and peripheral blood lymphocytes.
• the cells may be optionally stimulated with the human 0 ⁇ . receptor in vitro, or selected from donors which are known to have produced an immune response or donors who are HIV + but asymptomatic.
• the genetic information i.e., the human antibodies produced in the tissues in response to stimulation by ⁇ v -containing heterodimeric integrin antigen
• isolated from the donor cells can be in the form of DNA or RNA and is conveniently amplified by PCR or similar techniques.
• the genetic information is preferably converted into cDNA by reverse transcription prior to amplification.
• the amplification can be generalized or more specifically tailored. For example, by a careful selection of PCR primer sequences, selective amplification of immunoglobulin genes or subsets within that class of genes can be achieved.
• the component gene sequences are obtained, in this case the genes encoding the variable regions of the various heavy and light antibody chains, the light and heavy chain genes are associated in random combinations to form a random combinatorial library.
• the products can, after expression, be conveniently screened by biopanning with the selected human ⁇ v -containing receptor or, if necessary, by epitope blocked biopanning as described in more detail below.
• Fab fragments of mAbs such as B9
• B9 Fab fragments of mAbs
• single chain antibodies can also be used for cloning and screening.
• the present invention contemplates the use of Fab fragments or F(ab') 2 fragments to derived full-length mAbs directed against the human integrin ⁇ v ⁇ 3 or v ⁇ 5 receptor.
• fragments may be independently useful as protective and therapeutic agents in vivo against conditions mediated by the human ⁇ v -containing receptors or in vitro as part of a diagnostic for a disease mediated by the human ⁇ v -containing receptor, they are employed herein as a component of a reshaped human antibody.
• a Fab fragment contains the entire light chain and amino terminal portion of the heavy chain; and a F(ab') 2 fragment is the fragment formed by two Fab fragments bound by additional disulfide bonds.
• Human cv containing receptor binding monoclonal antibodies of the present invention provide sources of Fab fragments and F(ab') 2 fragments, which latter fragments can be obtained from combinatorial phage library [see, e.g., Winter et al., 1994, Ann. Rev. Immunol., 72:433-455 or Barbas et ai, 1992, Proc. Natl Acad. Sci. USA, 89:10164-10168 which are both hereby incorporated by reference in their entireties]. These Fab and F(ab') 2 fragments are useful themselves as therapeutic, prophylactic or diagnostic agents, and as donors of sequences including the variable regions and CDR sequences useful in the formation of recombinant or humanized antibodies as described herein.
• the mAb B9 or other antibodies described herein may contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR sequences, functional fragments, and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obtaining various altered antibodies which are characterized by the antigen binding specificity of the donor antibody.
• the present invention thus provides variable light chain and variable heavy chain sequences from the anti-human mAb B9 and sequences derived therefrom.
• the DNA and amino acid sequences of the heavy chain variable region of mAb B9 are reported illustrated by Fig. 1 A [SEQ ID NOS: 1 and 2].
• the nucleotide and amino acid sequences of the light chain variable region of mAb B9 are reported in Fig.
• nucleic acid sequences of this invention, or fragments thereof, encoding the variable light chain and heavy chain peptide sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incorporation of the resulting modified or fusion nucleic acid sequence into a plasmid for expression.
• silent substitutions in the nucleotide sequence of the framework and CDR-encoding regions can be used to create restriction enzyme sites which would facilitate insertion of mutagenized CDR (and/or framework) regions.
• These CDR-encoding regions may be used in the construction of reshaped human antibodies of this invention.
• variable heavy and light chain amino acid sequences may be constructed which encode the variable heavy and light chain amino acid sequences, and CDR sequences of the invention as well as functional fragments and analogs thereof which share the antigen specificity of the donor antibody.
• the isolated nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs can be used to produce altered antibodies, e.g., chimeric or humanized antibodies, or other engineered antibodies of this invention when operatively combined with a second immunoglobulin partner.
• nucleic acid sequences encoding portions of the altered antibody and antibodies described herein
• other such nucleic acid sequences are encompassed by the present invention, such as those complementary to the native CDR-encoding sequences or complementary to the modified human framework regions surrounding the CDR-encoding regions.
• Such sequences include all nucleic acid sequences which by virtue of the redundancy of the genetic code are capable of encoding the same amino acid sequences as provided in Figs. 1 A and 2A.
• An exemplary humanized light chain variable sequence is illustrated in Fig. 2C [SEQ ID NO: 9].
• An exemplary humanized heavy chain variable sequence is illustrated in Fig. IC [SEQ ID NO: 4].
• Figs. IA and 2A These heavy chain and the light chain variable regions have three CDR sequences described in detail in the murine sequences of Figs. IA and 2A.
• Other useful DNA sequences encompassed by this invention include those sequences which hybridize under stringent hybridization conditions [see, e.g., T. Maniatis et ai, 1982, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory pages 387 to 389] to the DNA sequences encoding the light and heavy chain variable regions of Figs. IA and 2A (also including Figs. IC and 2C for the synthetic human sequences) and which retain the antigen binding properties of those antibodies.
• hybridizing DNA sequences are at least about 18 nucleotides in length, i.e., about the size of a CDR.
• useful DNA sequences are those which are over 60%, preferably over 70% and more preferably over 80% or 90% homologous to the sequence or fragments of sequence of mAb B9.
• Altered Immunoglobulin Coding Regions and Altered Antibodies encode altered antibodies which include engineered antibodies such as chimeric antibodies, humanized, reshaped and immunologically edited human antibodies.
• a desired altered immunoglobulin coding region contains CDR-encoding regions in the form of Fab regions that encode peptides having the antigen specificity of the anti-human B9 antibody, preferably a high affinity antibody such as provided by the present invention, inserted into an acceptor immunoglobulin partner.
• the acceptor when the acceptor is an immunoglobulin partner, as defined above, it includes a sequence encoding a second antibody region of interest, for example an Fc region.
• Immunoglobulin partners may also include sequences encoding another immunoglobulin to which the light or heavy chain constant region is fused in frame or by means of a linker sequence. Engineered antibodies directed against functional fragments or analogs of the selected human ⁇ v subunit-containing integrin may be designed to elicit enhanced binding with the same antibody.
• the immunoglobulin partner may also be associated with effector agents as defined above, including non-protein carrier molecules, to which the immunoglobulin partner may be operatively linked by conventional means.
• Fusion or linkage between the immunoglobulin partners, e.g., antibody sequences, and the effector agent may be by any suitable means, e.g., by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde, and the like.
• suitable means e.g., by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde, and the like.
• linker sequences which simply provide for a desired amount of space between the second immunoglobulin partner and the effector agent may also be constructed into the altered immunoglobulin coding region.
• the design of such linkers is well known to those of skill in the art.
• signal sequences for the molecules of the invention may be modified to enhance expression.
• the reshaped human antibody having the signal sequence and CDRs derived from the mAb B9 heavy chain sequence may have the original signal peptide replaced with another signal sequence, such as the Campath leader sequence [Page, M. J. et al, 1991, BioTechnology, 9:64-68].
• An exemplary altered antibody a reshaped human antibody, contains a variable heavy and the entire light chain peptide or protein sequence having the antigen specificity of mAb B9 fused to the constant heavy regions C H ⁇ -C H 3 derived from a second human antibody.
• the engineered antibody of the invention may have attached to it an additional agent.
• the procedure of recombinant DNA technology may be used to produce an engineered antibody of the invention in which the Fc fragment or C H2 C H domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule (i.e., a polypeptide effector or reporter molecule).
• Another desirable protein of this invention may comprise a complete antibody molecule, having full length heavy and light chains, or any discrete fragment thereof, such as the Fab or F(ab') 2 fragments, a heavy chain dimer, or any minimal recombinant fragments thereof such as an F v or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor mAb B9.
• Such protein may be used in the form of an altered antibody, or may be used in its unfused form.
• an engineered antibody results.
• Engineered antibodies can comprise immunoglobulin (Ig) constant regions and variable framework regions from one source, e.g., the acceptor antibody or consensus sequences, and one or more (preferably all) CDRs from the donor antibody, e.g., the anti-human B9 antibody described herein.
• alterations e.g., deletions, substitutions, or additions, of the acceptor mAb light and/or heavy variable domain framework region at the nucleic acid or amino acid levels, or the donor CDR regions may be made in order to retain donor antibody antigen binding specificity or to reduce potential immunogenicity.
• Such engineered antibodies are designed to employ one (or both) of the variable heavy and/or light chains of the anti-human ⁇ v subunit-containing receptor mAb (optionally modified as described) or one or more of the below-identified heavy or light chain CDRs.
• the engineered antibodies of the invention are neutralizing, i.e., they desirably inhibit ligand binding to the v ⁇ 3 or ⁇ v ⁇ 5 receptor in vitro and in vivo in animal models of diseases mediated by these receptors, e.g., cancers.
• Such engineered antibodies may include a reshaped human antibody containing the human heavy and light chain constant regions fused to the human anti- ⁇ v ⁇ , 3 / v ⁇ 5 antibody functional fragments.
• a suitable human (or other animal) acceptor antibody may be one selected from a conventional database, e.g., the KABAT 7 database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
• a consensus sequence formed by all known human sequences in the database of a subgroup closest to that of the donor antibody may be used to supply the framework regions.
• a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
• a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
• heterologous framework and constant regions are selected from human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA and IgE.
• the Fc domains are not limited to native sequences, but include mutant variants known in the art that alter function. For example, mutations have been described in the Fc domains of certain IgG antibodies that reduce Fc-mediated complement and Fc receptor binding [see, e.g., A. R. Duncan et al., 1988, Nature, 332:563-564; A. R. Duncan and G. Winter, 1988, Nature, 332:738-740; M.-L. Alegre et al, 1992, J.
• the acceptor antibody need not comprise only human immunoglobulin protein sequences.
• a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding a non- immunoglobulin amino acid sequence such as a polypeptide effector or reporter molecule.
• a particularly desirable altered antibody is a humanized antibody containing all or a portion of the variable domain amino acid sequences of B9 and some portions of the donor antibody framework regions, or CDRs therefrom inserted onto the framework regions of a selected human antibody.
• This humanized antibody is directed against human v B 3 and ⁇ v ⁇ 5 receptors, among others.
• one, two or preferably three CDRs from the B9 antibody heavy chain and/or light chain variable regions are inserted into the framework regions of a selected human antibody or consensus sequence, replacing the native CDRs of that latter antibody or consensus sequence.
• variable domains in both human heavy and light chains have been engineered by one or more CDR replacements. It is possible to use all six CDRs, or various combinations of less than the six CDRs. For example, it is possible to replace the CDRs only in the human heavy chain, using as light chain the unmodified light chain from the human acceptor antibody. Still alternatively, a compatible light chain may be selected from another human antibody by recourse to the conventional antibody databases. The remainder of the engineered antibody may be derived from any suitable acceptor human immunoglobulin.
• the altered antibody thus preferably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use, e.g., treatment of diseases mediated by human receptors containing the ⁇ v subunit, or for diagnostic uses.
• variable domain amino acids may be further modified by changes in variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody (i.e., an analog). It is anticipated that heavy and light chain amino acids may be substituted by other amino acids either in the variable domain frameworks or CDRs or both. Particularly preferred is the immunological editing of such reconstructed sequences as illustrated in the examples herein.
• variable or constant region may be altered to enhance or decrease selective properties of the molecules of the instant invention by dimerization, binding to Fc receptors, or the ability to bind and activate complement [see, e.g., Angal et al, 1993, Mol. Immunol, 30:105-108; Xu et al, 1994, J. Biol. Chem., 269:3469-3474; Winter et al., EP 307,434-B], or as described above.
• Such antibodies are useful in the prevention and treatment of disorders mediated by these oC v -subunit containing integrins, as discussed herein.
• the resulting reshaped and engineered human, humanized and chimeric antibodies of this invention can be expressed in recombinant host cells, e.g., COS, CHO or myeloma cells, by resort to recombinant DNA technology using genetic engineering techniques. The same or similar techniques may also be employed to generate other embodiments of this invention.
• recombinant host cells e.g., COS, CHO or myeloma cells
• a conventional expression vector or recombinant plasmid is produced by placing these coding sequences for the altered antibody in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell.
• Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences, which can be derived from other known antibodies.
• a second expression vector can be produced having a DNA sequence which encodes a complementary antibody light or heavy chain.
• this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed.
• the heavy and light chain coding sequences for the altered antibody may reside on a single vector.
• a selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains.
• the transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention.
• the production of the antibody which includes the association of both the recombinant heavy chain and light chain is measured in the culture by an appropriate assay, such as ELISA or RIA.
• Similar conventional techniques may be employed to construct other altered antibodies and molecules of this invention.
• Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art.
• the conventional pUC series of cloning vectors may be used.
• One vector used is pUC19, which is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).
• any vector which is capable of replicating readily has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning.
• the selection of the cloning vector is not a limiting factor in this invention.
• the vectors employed for expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vectors.
• Preferred vectors include for example plasmids pCD or pCN.
• the vectors also contain selected regulatory sequences (such as CMV promoters) which direct the replication and expression of heterologous DNA sequences in selected host cells. These vectors contain the above described DNA sequences which code for the engineered antibody or altered immunoglobulin coding region. In addition, the vectors may incorporate the selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation.
• the expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR). Other preferable vector sequences include a polyadenylation (poly A) signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro).
• BGH bovine growth hormone
• betaglopro betaglobin promoter sequence
• replicons e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
• selection genes e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
• Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
• the present invention also encompasses a cell transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or altered immunoglobulin molecules thereof.
• Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. coli are used for replication of the cloning vectors and other steps in the construction of altered antibodies of this invention.
• Suitable host cells or cell lines for the expression of the engineered antibody or altered antibody of the invention are preferably mammalian cells such as CHO, COS, fibroblast cells (e.g., 3T3), and myeloma cells. More preferably a CHO or a myeloma cell is used. Human cells may be used, that enable the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., 1989, Molecular Cloning (A Laboratory Manual), 2nd edit., Cold Spring Harbor Laboratory (New York).
• Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs of the present invention [see, e.g., Pl ⁇ ckthun, A., 1992, Immunol. Rev., 730:151-188].
• the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form does not pose as great a concern, because Fabs are not normally glycosylated and can be engineered for exported expression thereby reducing the high concentration that facilitates misfolding. Nevertheless, any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability.
• bacterial cell If the molecule expressed by the bacterial cell was produced and exported in a properly folded form, that bacterial cell would be a desirable host.
• E. coli used for expression are well-known as host cells in the field of biotechnology.
• Various strains of B. subtilis, Streptomyces, other bacilli and the like may also be employed in this method.
• strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al, 1986, Genetic Engineering, 8:277-298 and references cited therein.
• the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the altered antibody of the invention from such host cell are all conventional techniques.
• the altered antibodies of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention.
• Yet another method of expression of reshaped antibodies may utilize expression in a transgenic animal, such as described in U. S. Patent No. 4,873,316, incorporated herein by reference.
• the engineered antibody is then examined for in vitro activity by use of an appropriate assay.
• Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the altered antibody to the human ⁇ v ⁇ 3 and v ⁇ 5 receptors. See, Example 3 below.
• other in vitro assays such as Example 9
• in vivo animal such as Examples 13 and 14
• models may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the altered antibody in the body despite the usual clearance mechanisms.
• B9 antibody is generated and expressed as described in detail in Example 1 1 below. V777. Therapeutic/Prophylactic Uses
• This invention also relates to a method of treating humans (or other mammals) experiencing symptoms related to diseases which are mediated by receptors such as v ⁇ 3 , ctyBs , otvB,, c. v ⁇ 6 , and ⁇ v ⁇ 8 , which comprises administering an effective dose of antibodies including one or more of the altered antibodies described herein or fragments thereof.
• the antibodies of this invention are useful for treating diseases wherein the underlying pathology is attributable to ligand which interacts with an v -containing receptor, such as the vitronectin receptor.
• these antibodies are useful as antitumor, anti-angiogenic, antiinflammatory and anti-metastatic agents, and are particularly useful in the treatment of atherosclerosis, restenosis, cancer metastasis, rheumatoid arthritis, diabetic retinopathy and macular degeneration.
• these antibodies are useful for treatment of conditions wherein loss of the bone matrix creates pathology.
• the instant antibodies are useful for the treatment of osteoporosis, hyperparathyroidism, Paget's disease, hypercalcemia of malignancy, osteolytic lesions produced by bone metastasis, bone loss due to immobilization or sex hormone deficiency.
• the therapeutic response induced by the use of the molecules of this invention is produced by the binding to the selected receptor, e.g., ⁇ v B or the others above mentioned, and thus subsequently blocking disease progression.
• the molecules of the present invention when in preparations and formulations appropriate for therapeutic use, are highly desirable for those persons experiencing disorders mediated by these human v -containing receptors. For example, longer treatments may be desirable when treating chronic diseases or the like.
• the dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient.
• the altered antibodies, antibodies and fragments thereof of this invention may also be used alone or in conjunction with other antibodies, particularly human or humanized or human antibodies reactive with other epitopes on the selected integrin receptor as prophylactic agents.
• the mode of administration of the therapeutic and prophylactic agents of the invention may be any suitable route which delivers the agent to the host.
• the altered antibodies, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously, or intranasally.
• Therapeutic and prophylactic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the altered antibody of the invention as an active ingredient in a pharmaceutically acceptable carrier.
• compositions for parenteral administration will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved in an pharmaceutically acceptable carrier, preferably an aqueous carrier.
• an aqueous carrier e.g. 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration).
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
• the concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1 %, to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
• a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg of an engineered antibody of the invention. Desirably the compositions may contain about 50 ng to about 80 mg of antibody, or more preferably, about 5 mg to about 75 mg of antibody according to this invention.
• a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 75 and preferably 5 to about 50 mg/ml of an engineered antibody of the invention. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art.
• the therapeutic and prophylactic agents of the invention when in a pharmaceutical preparation, be present in unit dose forms.
• the appropriate therapeutically effective dose can be determined readily by those of skill in the art.
• one dose of approximately 0.1 mg to approximately 20 mg per 70 kg body weight of a protein or an antibody of this invention should be administered parenterally, preferably i.v. or i.m. (intramuscularly).
• Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.
• the antibodies, altered antibodies or fragments thereof described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.
• the altered antibodies and monoclonal antibodies of this invention can be used in a combined therapy for the diseases described above with small molecule non-peptide antagonists of the selected integrin receptor.
• small molecule antagonists, the dosages and administration regimens are described in, e.g., International PCT patent publication No. WO96/00730, published January 11, 1996 and International PCT patent publication No. WO96/00574, published January 11, 1996, both incorporated by reference herein.
• Such combination therapy may involve administering an antibody of this invention to a patient for a short period, i.e., several months to six months, followed by chronic therapeutic treatment with the small molecule antagonists for a longer period of time.
• this embodiment of a method of treatment may involve alternating treatment periods of administering immunotherapy with the antibodies of this invention followed by small non-peptide antagonist treatments.
• Such combined therapeutic methods would employ the same dosages described above for the immunotherapy and the dosages specified in the above-cited applications for the non-peptide therapies.
• the altered antibodies and engineered antibodies of this invention may also be used in diagnostic regimens, such as for the determination of human ⁇ v -containing receptor- mediated disorders or tracking progress of treatment of such disorders.
• diagnostic regimens such as for the determination of human ⁇ v -containing receptor- mediated disorders or tracking progress of treatment of such disorders.
• these altered antibodies may be conventionally labeled for use in ELISAs and other conventional assay formats for the measurement of human c v subunit-containing receptor levels in serum, plasma or other appropriate tissue or the release by human cells in culture.
• the nature of the assay in which the altered antibodies are used are conventional and do not limit this disclosure.
• EXAMPLE 1 PURIFICATION OF ⁇ . ⁇ 3 , ⁇ AND ⁇ v ⁇ , RECEPTORS
• the human ⁇ v ⁇ 3 protein receptor and other protein receptors were purified from human placenta as follows. Placentas were frozen immediately after birth, then partially thawed and cut into small chunks which were ground to fine pieces using a commercial meat grinder. Usually five to ten placentas were ground at one time; the pieces were placed into 50 ml centrifuge tubes (6 tubes per placenta) and stored frozen at -20E C until use.
• Anti-oU_ 3 mAb (LM609) was purified from mouse ascites purchased from Chemicon International, Inc. (Temecula, CA). Monoclonal antibodies 23C6 or D12 were purified from hybridoma media.
• mAbl ⁇ were purchased from Becton Dickinson. LM609 or 23C6 or D12 (50 mg), P1F6 (25 mg), and mAbl ⁇ (25 mg) were immobilized on AffiGel 10 (BioRad) at 5 mg of mAb/ml of resin following the manufacturer's instruction. In order to remove the nonspecific binding proteins, 20 ml of AffiGel 10 was treated with 1 M Tris HCI pH 7.5 and packed in an Econo Column.
• the immobilized mAb's were packed in EconoColumn (BioRad), 10 ml column for LM609 or 23C6 or D 12, 5 ml one for PlF ⁇ and 5 ml one for mAb 16.
• the columns were connected in tandem: the first column containing AffiGel 10 for nonspecific binding, the second column containing v ⁇ 3 mAb, the third column containing o ⁇ mAb and the fourth column containing P1F6 (the ⁇ v ⁇ 5 ) mAb.
• the columns were equilibrated with buffer T (50 mM TrisHCl, pH 7.5, 0.1 M NaCl, 2 mM CaCl, 1 % octyl glucoside) in the coldroom.
• the ground placenta (9 tubes) was partially thawed and dispersed thoroughly using spatula in buffer T+6% octyl glucoside (final concentration of OG was 3%). The mixture was stored for 5 hours or overnight at 4 C.
• the bulky solution was transferred to 250 ml centrifuge bottles and centrifuged at 13,000 rpm for one hour.
• the clear supernatant was transferred to 50 ml centrifuge tubes and centrifuged at 20,000 rpm for one hour.
• the clear supernatant was combined and loaded with the flow-rate of 30 ml/hour to the columns arranged and pre-equilibrated in buffer T in tandem mode as described above. At the end of loading, the columns were washed with >250 ml of buffer T.
• the eluted integrin solutions ( 25 ml) were concentrated to 1 ml using Aquaside III (Calbiochem) in a dialysis bag of 5000 cut off.
• the concentrated integrins were dialyzed overnight against buffer T. The final yield was approximately 1 mg for each integrin per placenta.
• EXAMPLE 2 GENERATION OF MURINE MONOCLONAL ANTIBODIES
• Murine mAbs with anti-a v B 3 activity were generated by classical hybridoma technology according to Lane et al, 1986, Methods in Enzymol., 121: 183. Generally, 20-50 ⁇ g of ⁇ v ⁇ . receptor was administered ip, sc, and iv to two Balb/c mice. Sera from the immunized animals were tested for their anti- v ⁇ 3 binding and neutralizing activity in assays of Examples 3, 4 and 5 below. Mouse spleen from mice showing positive sera was fused with a mouse myeloma cell SP2 according to the procedures of Lane et al, cited above. Seventeen resulting hybridoma cell lines, secreting potential anti-human ⁇ v B3 protein antibodies were obtained. These anti- ⁇ v B 3 mAbs were generated and isolated from culture by conventional methods and tested in assays of the following examples.
• Table I is a summary of the data collected from Examples 3-10 below on the murine mAb B9, the murine mAb LM609 of the prior art and other murine mAbs of this invention.
• the data showed that mAb B9 was a mAb with favorable activity profile.
• the mAb B9 that functioned adequately in these tests was then selected for humanization as described in Example 1 1 , and further tested in animal models of Examples 13 and 14.
• the B9 mAb has minimal cross-reactivity with rabbit (not pursuable for in vivo studies), therefore human and primate models of restenosis, angiogenesis or atherosclerosis are applicable for testing efficacy.
• EXAMPLE 3 ELISA BINDING ASSAY WITH oU_ 3 and ⁇ ⁇ Binding of the various antibody constructs to purified human placenta ⁇ B receptor protein and v ⁇ s receptor protein as antigens (receptor either bound to the plate or to the beads via biotm-avidin) was measured in a standard solid phase ELISA
• Antigen diluted in 0 1 M CO pH 9 2 was adsorbed onto polystyrene round-bottom microplates (Dynatech, Immunolon II) for 18 hours Wells were then washed one time with phosphate buffered saline (PBS) containing 0 05% Tween 20 Antibodies (50 ⁇ l/well) were diluted to varying concentrations in PBS/0 05% Tween 20 and added to the antigen coated wells for two hours at room temperature Plates were washed four times with PBS containing 0 05% Tween 20, using a Titertek 320 microplate washer, followed by addition of HRP-anti-mouse IgG (100 ⁇ l/well) diluted 1 10,000
• o-phenylenediamine dihydrochlo ⁇ de (1 mg/ml) was added and plates were incubated an additional 10 minutes The reaction was stopped by addition of 0 1M NaF and absorbance read at 450 nm using a Dynatech MR 7000 ELISA reader EXAMPLE 4 ELISA BINDING ASSAYS WITH , ⁇ a& and ⁇ structuralh ⁇ , and o ⁇ _,
• MAbs positive in the assay of Example 3 were screened using the same protocols except that the antigen was another human receptor, o ⁇ B] or 01 ⁇ These assays were run to determine selectivity for the heterodimenc antigen ⁇ v B and c. v ⁇ ; as opposed to selectivity for a ⁇ subunit only The results of these assays are reported in Table I above for all mAbs of Example 2 and for LM609
• the secondary antibody Goat Anti-Mouse IgG,M,A is labelled with FITC 1 25/PBS- 0 2% BSA-0 1 % NaN 3 (Sigma F1010 lot #045H8822) and hold cold in the dark until read by FLOW
• a BIAcore analysis (Pharmacia) was performed to measure binding affinity of mAb B9 (6nM) with immobilized ⁇ v B and ⁇ v ⁇ 5
• the interactions of these receptors with B9 were studied using BIAcore technology by immobilization of the receptors onto the sensor surface, and passing solutions of the mAb over this surface
• the receptors was immobilized by inserting it into a phospholipid vesicle and producing a hybrid bilayer membrane on a hydrophobic sensor surface.
• the k ass (rate of association) and the K diss (rate of dissociation) are measurements which calculate the affinity of the mAbs against the ⁇ v B3 receptor (K D in nM).
• This Biacore data indicates that the B9 mAbs have a higher affinity for the v B 3 receptor than does the DI 2 mAb.
• Smooth muscle cell (SMC) migration from the media into the wound area to initiate growth of the neointima is an essential remodeling response following vascular injury. Inhibition of SMC migration attenuates neointima formation.
• Vascular SMC migration is mediated via the human ⁇ v B receptor, which is expressed in VSMC and upregulated following vascular injury.
• Osteopontin a ligand of the human v B3 receptor, is upregulated following angioplasty and promotes VSMC migration via the integrin. This experiment was performed to demonstrate the ability of an antibody to human ⁇ v B to inhibit VSMC migration in vitro.
• DMEM Difco's minimal essential medium
• BSA at a concentration of 2.5 - 5.0 x 10 6 cells/mL, and were pretreated with test antibody at various concentrations for 20 minutes at 20E C.
• the solvent alone was used as control.
• 0.2 mL of the cell suspension was placed in the upper compartment of the chamber.
• the lower compartment contained 0.6 mL of DMEM supplemented with 0.2% BSA. Incubation was carried out at 37E C in an atmosphere of 95% air/5% C0 2 for 24 hours.
• the non-migrated cells on the upper surface of the filter were removed by gentle scraping.
• the filter was then fixed in methanol and stained with 10% Giemsa stain. Migration was measured either by a) counting the number of cells that had migrated to the lower surface of the filter or by b) extracting the stained cells with 10% acetic acid followed by determining the absorbance at 600 nM.
• EXAMPLE 10 HEK293 CELL ADHESION TO DETERMINE INHIBITION OF ADHESION.
• Human embryonic kidney cells (HEK293 cells) were obtained from ATCC (Catalog
• the growth medium was supplemented with 450 ⁇ g/ l Geneticin (G418 Sulfate, GIBCO-BRL, Bethesda, MD). The cells were maintained in selection medium until the colonies were large enough to be assayed.
• Corning 96-well ELISA plates were precoated overnight at 4E C with 0.1 ml of human vitronectin (0.2 ⁇ g/ml in RPMI medium). At the time of the experiment, the plates were washed once with RPMI medium and blocked with 3.5% BSA in RPMI medium for 1 hour at room temperature. Transfected 293 cells were resuspended in RPMI medium, supplemented with 20 mM Hepes, pH 7.4 and 0.1 % BSA at a density of 0.5 x 10 6 cells/ml. 0.1 ml of cell suspension was added to each well and incubated for 1 hour at 37E C, in the presence or absence of various ⁇ v ⁇ 3 antagonists.
• VH and VL regions were synthesized by combining the framework regions of the human V region subgroup consensus sequences together with the CDR regions of B9.
• Molecular modeling of B9 using known crystal structures reveals certain VH and VL framework residues that can make contact with CDR residues, and thereby influence their conformation. Such framework residues can therefore directly contribute to the formation of a particular antigen specificity and associated binding affinity.
• N-terminal amino acid microsequencing was performed on B9 heavy and light chains.
• the N-terminal VH sequence, QVQLQQSGAELMKPGA [SEQ ID NO :16] and Vk sequence, DIQMTQTTSSLXAXL [SEQ ID NO: 17 ] were used to design degenerate PCR primers that could direct PCR amplification of the unknown VH and Vk regions, respectively. These degenerate primers were designed such that the amplified V-regions would nevertheless encode authentic N-termini.
• PCR amplification of the RNA/DNA hybrid was carried out for 25 cycles using a mouse kappa reverse primer SBA0352: 5' GCA CCT CCA GAT GTT AAC TGC 3' [SEQ ID NO. 20] and a degenerate forward primer based on the Vk N-terminal protein sequence SBA9258: 5' GCT ACC GGT GTC CAC TCC GA(C/T) AT(A/C/T) CA(A/G) ATG ACN CA 3' [SEQ ID NO. 21 ].
• the PCR DNA was analyzed on a 0.8% agarose gel. PCR inserts of the appropriate size, ⁇ 400bp, were sequenced by a modification of the Sanger method.
• pCDHCAGE is a variant of the plasmids pCDN [A. Nambi et al, 1994, Mol. Cell. Biochem.. 131 :75-85] and F9HZLC1-1- pCN [International patent publication WO94/05690].
• pCDN variant plasmid vectors contain, in general, a beta lactamase gene, a SV40 origin of replication, a cytomegalovirus promoter sequence, a selected humanized light chain, a poly A signal for bovine growth hormone, a beta globin promoter, a neomycin resistance gene, and another BGH sequence poly A signal in a pUCl 9 background.
• pCDHCAGE further contains the CAMPATH immunoglobulin leader sequence in which a unique Agel site had been introduced at the 3' end, a linker sequence followed by uninterrupted heavy chain coding sequences commencing at a unique Apal site at the 5' end of the human CHI region through to the end of the human CH3 region.
• a synthetic humanized light chain variable region was designed using a human light chain subgroup I variable region consensus framework as described by Kabat and the B9 light chain CDRs as described previously.
• One murine framework amino acid substitution which might influence CDR presentation was introduced (see asterisk in Fig 2C; [SEQ ID NOS. 9 and 10]).
• Four overlapping synthetic ohgonucleotides were generated with the following sequences:
• SBB0030 5'TTT TTG GCT ACC GGT GTC CAC TCC GAT ATT CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCC TCT GTG GGA GAC AGA GTC ACC ATC ACT TGC AGG 3' [SEQ ID NO.22]; SBB0031:5'TAA TGT TGA TGT GTA GTA GAT CAG AAG
• SBB0032 5'CTG ATC TAC TAC ACA TCA ACA TTA CAC TCA GGA GTC CCA TCA AGG TTC AGT GGC AGT GGG TCT GGA ACA GAT TAT ACT CTC ACC ATT AGC AGC CTG CAG 3' [SEQ ID NO.24];
• This synthetic Vk region was then PCR amplified using primers SBB0034: 5' TTT TTG GCT ACC GGT GTC CAC TCC GAT ATT CAG 3' [SEQ ID NO. 26] and SBB0035: 5' ACG GGT ACC TCC ACC GAA CGT CCA AGG 3' [SEQ ID NO. 27] and ligated into the pCR2000 cloning vector (TA cloning Kit, Invitrogen, Cat. No. K2000-01), and isolated after a Agel, Kpnl restriction digest. This DNA fragment was ligated into the vector pCNLCAGE which had previously been restriction digested with Agel and Kpnl.
• pCNLCAGE is a variant of the plasmids pCDN [ A. Nambi et al, 1994, Mol. Cell. Biochem.. 131 :75-85] and F9HZLCl-l-pCN [International patent publication WO94/05690].
• These pCDN variant plasmid vectors contain, in general, a beta lactamase gene, a SV40 origin of replication, a cytomegalovirus promoter sequence, a selected humanized light chain, a poly A signal for bovine growth hormone, a beta globin promoter, a neomycin resistance gene, and another BGH sequence poly A signal in a pUC19 background.
• pCNLCAGE further contains the CAMPATH immunoglobulin leader sequence in which a unique Agel site had been introduced at the 3' end, a linker sequence followed by uninterrupted light chain coding sequences commencing at a unique Kpnl site in the middle of the human Jk region through to the end of the human Ck region (i.e., the light chain constant region of the k isotype, as opposed to the lambda isotype).
• the resulting gene codes for a complete humanized light chain, comprising the initiation codon and the end of the Ck domain.
• This expression construct is called pCNB9HZLCl -0, and when cultured in a suitable host cell, produces a complete human k chain with the humanized B9 light chain V-region B9HZLC1 -0 (Fig 2C ;[SEQ ID NOS. 9 and 10]).
• the humanized HuB9 heavy and light chains were expressed in COS cells essentially as described in Current Protocols in Molecular Biology (edited by F. M. Ausubel et al, 1988, John Wiley & Sons, vol. 1, section 9.1). Briefly described, the COS cells were co-transfected with 10 ⁇ g of each plasmid. On day 1 after the transfection, the culture growth medium was replaced with a serum-free medium which was changed on day 3. The serum-free medium was a proprietary formulation, but satisfactory results are obtained using DMEM supplemented with ITSTM Premix (insulin, transferrin, selenium mixture - Collaborative Research, Bedford, MA) and 1 mg/ml BSA.
• ITSTM Premix insulin, transferrin, selenium mixture - Collaborative Research, Bedford, MA
• the mAb was isolated and prepared from the day 3 + day 5 conditioned medium by standard protein A affinity chromatography methods (e.g., as described in Protocols in Molecular Biology) using, for example, Prosep A affinity resin (Bioprocessing Ltd., UK).
• dhfr CHO cells as previously described [P. Hensley et al., cited above]. Briefly, a total of 30 ⁇ g of linearized plasmid DNA (15 ⁇ g each of the heavy or light chain plasmids) is electroporated into lxlO 7 cells. The cells are initially selected in nucleoside-free medium in 96 well plates. After three to four weeks, media from growth positive wells is screened for human immunoglobulin using the ELISA assay of Example 3. The highest expressing colonies are expanded and selected in increasing concentrations of methotrexate for amplification of the transfected vectors. The antibody is purified from conditioned medium by standard procedures using protein A affinity chromatography
• the concentration and the antigen binding activity of the eluted antibody are measured by the ELISA assays of Examples 3 and 4.
• the antibody containing fractions are pooled and further purified by size exclusion chromatography.
• a second construct has a light chain based on the human REI consensus framework to provide an alternative light chain in the event of unstable expression in humanized B9 production cell lines.
• This variant differs from B9HZLC1-0 by only two conservative amino acid interchanges, L73F and F83I (numbering convention of Kabat).
• a third B9 humanized light chain variant was created that differed from B9HZLC1-0 by the single amino interchange K103N (numbering convention of Kabat). This variant was created by site directed mutagenesis of the B9HZLC1-0 intermediate (Fig. 4 [SEQ ID NOS 13 and 14]) and cloned into pCNLCAGE as described above. The resulting humanized V region was that of B9HZLC1 -1 of Fig. 7 [SEQ ID NOS: 30 and 31].
• the SCID model in which human skin is grafted on the SCID mouse, which serves as a source of angiogenic neovascularization, and subsequently accepts human tumor (tumor growth supported by human vasculature) is utilized for efficacy testing of the B9 mAbs and HuB9 antibodies. Inhibition of tumor growth indicates that B9 mAbs (human anti- ⁇ v ⁇ 3 and anti- ⁇ v ⁇ 5 positive, murine anti- ⁇ v ⁇ _3 negative) play a role in the inhibition of ⁇ v B . 3 dependent angiogenesis.
• Antibodies of this invention are also tested for in vivo activities in other mammalian models of cancer metastasis, rheumatoid arthritis and atherosclerosis, e.g., baboon models.
• EXAMPLE 14 MOUSE TUMOR XENOGRAFT MODEL WITH HT29
• the cells were injected subcutaneously.
• the s.c. tumor is a more vigorous system than the i.p. tumor described above. See Fig. 9.
• EXAMPLE 15 HUMAN AORTA RING ANGIOGENESIS MODEL This in vitro assay is performed to measure anti-angiogenic activity. Human thoracic aorta is extracted and sectioned into roughly 1 mm sections and placed into 24 well culture plates that contain a three-dimensional matrix. Endothelial cells form vessel-like capillaries branching off from the aorta. B9 mAbs are introduced into this system and their effect on angiogenesis is studied.

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